Robert Luedtke - US grants

This grant application is the molecular pharmacology portion of an Investigator Initiated Interactive Research Project designed to identify pharmacotherapeutic agents that can be used in the treatment of cocaine addiction. The strategy of this research initiative is three-fold. I. Medicinal Chemistry: A panel of 50-75 novel benzamide derivatives of 2,3-dimethoxy-N-(p-flurobenzyl)piperdin-4-yl benzamide (MBP) and 2,3 - dimethoxy-N-(9-p-flurobenzyl)-azabicyclo[3.3.1]nonan-3beta-yl benzarnide (MABN), which are known to be high-affinity, nonselective antagonists for D2 and D3 receptors, will be synthesized. ll. Pharmacology: Genetically engineered eukayotic cells, expressing high levels of D2-like dopamine (D2, D3 and D4) receptors, will be used to characterize the pharmacologic selectivity of each benzamide derivative for the three D2-like dopamine receptor subtypes. Ill. Behavioral Studies: Those benzamide derivatives exhibiting the greatest selectivity will be used 1) to determine their efficacy for antagonizing the reinforcing effects of cocaine self-administration in primates using behavioral paradigms, 2) to investigate the molecular properties of the D2-like neurotransmitter binding sites using affinity labeling techniques, and 3) to explore the potential for using receptor subtype selective irreversible antagonists as a therapeutic tools for drug abuse rehabilitation. Although cocaine's reinforcing effects are known to be mediated through dopamine neurotransmission, it is not clear which of the dopamine receptor subtypes play a predominant role in reinforcement. One obstacle to defining which dopamine receptor subtype(s) are involved in reinforcing effects is the lack of high-affinity, selective antagonists for each of the receptor subtypes. The experimental strategy for this proposal is to 1) develop a genetically engineered recombinant baculovirus virus that can be used to infect Sf9 cells for the expression of D2, D3 and D4 receptors, 2) use radioligand binding techniques to define the pharmacologic selectivity of a panel of novel benzamides for D2, D3 and D4 receptors expressed in Sf9 cells, and 3) prepare and characterize affinity labeling reagents that can be used a) in concert with dopamine receptor subtypes expressed in Sf9 cells to determine the position and orientation of benzamides within the neurotransmitter binding sites of D2, D3 and D4 dopamine receptors, and b) to explore the feasibility of using irreversible receptor blocking agents as a therapeutic tool in the treatment in cocaine abuse.

Therapeutics for Cocaine Dependence: Natural Products Stimulation of dopamine receptor subtypes in the mesolimbic area of the brain appears to be directly associated with the cocaine-seeking behaviors. Therefore, the development of pharmacologic agents that selectively interact with dopamine receptor subtypes has the potential for yielding a pharmacotherapy for the various stages for cocaine addiction, including priming, reinforcement, craving, relapse and/or euphoria. Recent studies suggest that D-2 like dopamine receptor stimulation may mediate the incentive to seek further cocaine reinforcement, while D1-like dopamine receptor stimulation may attenuate reinforcement (Self et al., 1996). Natural plant products have served in the past as a rich source for the development of pharmacotherapeutic agents. Therefore, it is probably that natural compounds having dopaminergic activity can be identified by testing extracts of natural products. The goal of this grant application is to identify the extracts of natural products which have activity at D1- like and/or D2-like dopamine receptors. Initial pharmacologic characterization of the active extracts to determine the pharmacologic selectivity of the extract for each of the five D1-like (D1a and D1b) and D2-like (D2, D3, and D4) dopamine receptor subtypes and to determine whether the active component is an antagonist, agonist, partial agonists or inverse agonist at each of the D1-like and D2-like dopamine receptor subtypes. Purification and structural identification active components. The final goal of this application is to identify novel lead compounds that can be used for the development of natural, semi- synthetic or synthetic compounds that are either D1-like (D1a and D1b) dopamine receptor selective agonists or partial agonists or D3 dopamine receptor subtype selective antagonists.

DESCRIPTION (Provided by applicant): A variety of neurological and neuropsychiatric disorders appear to be due to disturbance of the dopaminergic system. Since recent studies indicate that Di-like and D4 dopamine receptor selective antagonists are not effective antipsychotics, there is a renewed focus on D3 dopamine receptors as a target for antipsychotic drugs used in the treatment of schizophrenia. In addition, recent studies suggest that the D3 dopamine receptor subtype might be an important target for the development of agents for pharmacotherapeutics that could be used in the rehabilitation of individuals who abuse cocaine. However, it has been difficult to develop selective D3 receptor compounds that can be used for experimental or clinical studies on the role of the D3 dopamine receptor subtype in neuropsychiatric disorders or drug abuse because the D2 and D3 dopamine receptors have a high degree of amino acid sequence homology within the transmembrane spanning regions, which construct the neutotransmitter binding site. In collaborative studies, our laboratory has identified a series of structurally related compounds that range from 5- to 50-fold selective for the LB compared to the D2 dopamine receptor subtype. The experiments described in this proposal are designed to identify the amino acid residues within the D2 and LB dopamine receptor binding sites that directly interact with the currently available LB selective compounds. This will be accomplished by preparing mutant receptors structurally related to the D2 and LB dopamine receptor subtypes to precisely define the position of the pharmacophore within the neurotranamitter binding site. The results of these studies will 1) provide information on how our current LB dopamine receptor selective compounds bind to the neumtransmitter binding sate and 2) provide additional structural information that will assist us in the design of novel compounds with increased selectivity for LB dopamine receptors

? DESCRIPTION (provided by applicant): While dopaminergic pathways have been clearly implicated in the reinforcing aspects of reward, the precise role of the dopamine receptor subtypes in motivation for drug seeking behaviors, reinstatement, drug craving and relapse remains unclear. The hypothesis for this application is that arylamide phenylpiperazine analogs, that bind with high affinity at D3 dopamine receptors and varying D3 vs. D2 receptor selectivity, can be used safely for the treatment of cocaine abuse in the context of a drug rehabilitation program. Based upon the information gained from our previous studies, we propose new synthetic strategies to identify compounds with the desired pharmacological profiles and pharmacokinetic (PK) properties appropriate for the treatment of cocaine abuse in humans by 1) modifying the orthosteric pharmacophore (phenylpiperazine moiety) and 2) integrating information obtained from binding and functional studies, as well as findings from adsorption, distribution, metabolic, elimination (ADME) studies and PK analysis into the synthetic design. Select compounds will then be tested in rats to evaluate their ability to attenuate cocaine-associated behaviors. This research has the potential to lead to a new treatment strategy for cocaine dependence and psychostimulant abuse-related behaviors. Aim 1. Synthesis of novel D3 receptor selective compound with increased efficacy. We propose to modify the phenylpiperazine orthosteric pharmacophore by synthesizing panels of novel arylamide homopiperazine, imidazolidine, aza-tropane and aza-granatane analogs. Aim 2. Evaluation of binding and functional selectivity. The affinity of analogs described in Aim 1 will be determined at D1 dopamine, D2-like (D2, D3 and D4) dopamine, sigma 1 and 2 receptors using radioligand binding techniques. In addition, the intrinsic efficacy of our novel compounds will be evaluated using multiple in vitro functional assays including, a) adenylyl cyclase inhibition, b) mitogenesis c) MAPK/pERK pathway activation and d) ¿-arrestin binding. Compounds will also be tested in vivo for cFos activation, effects on spontaneous locomotion and effects on rotarod performance. Aim 3. Evaluation of selected compounds for ADME and pharmacokinetic properties. Based upon binding and functional selectivity profile (Aim 2), a select panel of D3 receptor selective compounds will be evaluated using a battery of in vitro ADME and in vivo PK profiling assays. Aim 4. Evaluate the effects of the newly synthesized compounds on cocaine reinforcement, motivation and motor function. A select panel of novel compounds (Aims 1-3) will be evaluated for their effects on cocaine self-administration under low and progressive ratio reinforcement schedules and on reinstatement of extinguished cocaine seeking behavior.

Abstract The prevalence of Alzheimer's disease (AD) and the associated financial and caregiver burden is projected to escalate dramatically unless disease-modifying treatments are discovered. Our research is focused on addressing this urgent unmet medical and societal need through the discovery and development of pharmacotherapies capable of averting or delaying the progression of AD. Brain inflammation initiated by chronic oxidative-nitrosative stress is a proven component of the pathogenic cascade leading to mild cognitive impairment (MCI) and AD. When surplus inflammatory nitric oxide and superoxide molecules combine they form the brain-impairing reactive species peroxynitrite. This perpetuates inflammation resulting in the progressive neurodegeneration observed in AD. Our innovative approach consists of managing two processes associated with inflammatory disease progression. The first involves interrupting the cycle of peroxynitrite generation by suppressing unsafe elevations in nitric oxide triggered by oxidative stress, and the second involves enhancing resilience to and recovery from inflammatory insults by facilitating the secretion of brain-derived neurotrophic factor (BDNF). A single stress-activated chaperone protein is mechanistically capable of both mediating BDNF secretion and regulating nitric oxide levels under proinflammatory conditions. Preliminary studies have identified chemotype starting points for further CNS drug development which have the desired dual functional selectivity profile for this target receptor. Our plan is to optimize the CNS drug-like properties of these functionally selective chemotypes with the goal of developing medicines that can significantly modify the course of MCI/AD.